Bta-miR-101对牛睾丸支持细胞增殖、凋亡及分泌的影响  

Effects of bta-miR-101 on Proliferation,Apoptosis and Secretion of Bovine Testicular Sertoli Cells

作　　者：虎巧燕 翟相钦 李一丹 韩家乐 雷初朝[1] 党瑞华[1] HU Qiaoyan;ZHAI Xiangqin;LI Yidan;HAN Jiale;LEI Chuzhao;DANG Ruihua(College of Animal Science and Technology,Northwest A&F University,Yangling 712100,China)

机构地区：[1]西北农林科技大学动物科技学院,杨凌712100

出　　处：《畜牧兽医学报》2024年第3期1040-1051,共12页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家肉牛牦牛产业技术体系项目(CARS-37);陕西省秦创原科学家+工程师项目(2022KXJ-112);中央引导地方项目(K3030922098)。

摘　　要：旨在探究bta-miR-101对牛睾丸支持细胞增殖、凋亡及分泌的影响。本研究采集3日龄(n=3)与13月龄(n=3)健康公牛的睾丸组织,构建安格斯牛组织表达谱,验证bta-miR-101在两个时期的差异表达。利用在线工具(TargetScan、miRTarBase、miRDB及miRWalk)预测miR-101的靶基因,并利用KOBAS对其进行GO和KEGG通路富集分析。将miR-101的模拟物与抑制物分别转染至牛睾丸支持细胞中,利用RT-qPCR、Western Blotting以及CCK-8等技术检测细胞增殖、凋亡及分泌相关基因表达量以及细胞增殖系数。通过4种在线软件预测到26个bta-miR-101的共同靶基因,GO和KEGG富集分析发现这些基因主要富集在核质、蛋白质结合、JAK-STAT的受体信号通路上,这些信号通路与细胞分化、细胞周期、细胞凋亡等生物过程有着不同程度的关联。RT-qPCR结果显示,bta-miR-101在牛初生期睾丸组织中的表达量显著高于性成熟时期(P<0.05),与实验室前期测序结果一致。转染miR-101的模拟物与抑制物后,发现相较于对照组,miR-101 mimics可以促进增殖相关基因在mRNA和蛋白水平的表达量,并显著升高支持细胞的增殖系数,同时抑制凋亡基因在mRNA和蛋白水平的表达量(P<0.05);miR-101 inhibitor可以抑制增殖相关基因在mRNA和蛋白水平的表达量,同时促进凋亡基因在mRNA和蛋白水平的表达量(P<0.05)。在细胞分泌方面,miR-101对分泌标志基因GDNF、BMP4的转录有影响,但对雄激素结合蛋白的分泌影响不显著。综上所述,miR-101促进牛睾丸未成熟支持细胞的增殖并抑制其凋亡,对雄激素结合蛋白的分泌无显著影响。The study aimed to explore the effects of bta-miR-101 on bovine testicular sertoli cells’proliferation,apoptosis and secretion.In this study,testicular tissues of healthy bulls(n=3)aged 3 days and 13 months were collected to construct the expression profile of Angus cattle tissues.The differences in bta-miR-101 expression were verified between the two periods.Online tools(TargetScan,miRTarBase,miRDB,and miRWalk)were used to predict the target gene of miR-101,and KOBAS was used for enrichment analysis of GO and KEGG pathways.The mimics and inhibitors of miR-101 were transfected into bovine testicular sertoli cells.Cell proliferation,apoptosis,secretion-related gene expression,and cell proliferation coefficient were detected by RT-qPCR,Western Blotting,and CCK-8 techniques.There were 26 common target genes for bta-miR-101 predicted by 4 online tools.GO and KEGG enrichment analysis showed that these genes were mainly enriched in the nucleoplasm,protein-binding,and JAK-STAT receptor signaling pathways,which were associated with cell differentiation,cell cycle,apoptosis,and other biological processes to varying degrees.The results of RT-qPCR showed that bta-miR-101 expression was significantly higher in testis tissue at birth than at sexual maturity(P<0.05),consistent with previous laboratory sequencing results.After transfection of miR-101 mimics and inhibitors,it was found that miR-101 mimics could promote the expression of proliferation-related genes at mRNA and protein levels,significantly increase the proliferation coefficient of sertoli cells,and inhibit the expression of apoptosis genes at mRNA and protein levels(P<0.05);miR-101 inhibitors inhibited the expression of proliferation-related genes at mRNA and protein levels and promoted the expression of apoptotic genes at mRNA and protein levels(P<0.05).In terms of cell secretion,miR-101 had an effect on the transcription of secretion marker genes GDNF and BMP 4,but had no significant effect on the secretion of androgen-binding proteins.In conclusion,miR-101 pr

关 键 词：牛睾丸支持细胞 miR-101 增殖 凋亡 

分 类 号：S823.3[农业科学—畜牧学]